Compositions and methods of sensitizing methicillin resistant Staphylococcus aureus to beta-lactam antibiotics

ABSTRACT

Contemplated methods and compositions further increase susceptibility of sensitized MRSA against various antibiotic drugs. Most preferably, the MRSA is already sensitized with a galloylated catechin (e.g., ECG), and further sensitization is achieved by exposure to a non-galloylated catechin (e.g., EC), and most preferably the corresponding non-galloylated catechin.

FIELD OF THE INVENTION

The field of the invention is antibiotic treatment compositions andmethods, and particularly treatment of methicillin resistantStaphylococcus aureus (MRSA) that include synergistic combinations ofselected catechins.

BACKGROUND OF THE INVENTION

Epigallocatechin gallate (EGCG) and other tea catechins have previouslybeen used as antibacterial agents, and examples for such activity arewell known in the art (see e.g., Jpn. J. Bacteriol. (1991); 46:839-845). However, to exhibit significant antibacterial effect, dosagesare generally very high and are therefore typically not achieved inphysiological conditions or in vivo. Similar high concentrations werereported by Shimamura in U.S. Pat. No. 5,358,713 using selectedcatechins to prevent transmission of MRSA to another person.

To reduce minimum inhibitory concentration of certain antibiotic drugs,antibiotic drugs were combined with certain catechins, and selectedcatechins were reported to be sensitizing agents for amikacin,chloramphenicol, and various beta-lactam antibiotics as described byShimamura in U.S. Pat. No. 5,807,564. Similarly, rose polyphenols werereported to reduce the minimum inhibitory concentration of variousbeta-lactam antibiotics (see e.g., Microbiol Immunol. 2004;48(1):67-73.). However, and at least for some of the catechins, theeffective concentrations require were still relatively high. In otherattempts to treat MRSA infections, selected catechins were also combinedwith specific theaflavin compounds as described in EP 0 761 226 toproduce an antimicrobial composition. Unfortunately, the samedifficulties remained with respect to certain catechins. In yet furtherknown combinations of selected catechins, Morree et al disclosed in U.S.Pat. No. 6,652,890 a mixture of EGC and EC that was effective ininhibiting NADH oxidase of neoplastic cells that express NOX to therebytreat cancer. However, the effect of such combination on bacteria wasnot reported in that reference.

In still further known attempts to use catechins as antibiotic drugs,various catechins were chemically modified as described by Stapleton etal in WO 2005/034976. While such modified catechins provided remarkableantibacterial effect as compared to their unmodified counterparts,synthesis and purification increases cost, and administration to humanhas not yet been proven safe and effective.

Thus, while numerous compositions and methods for eradicating and/orsensitizing MRSA to various antibiotics are known in the art, all oralmost all of them suffer from one or more disadvantages. Therefore,there is still a need for improved pharmaceutical agents for treatmentand/or chemoprevention of infections with MRSA.

SUMMARY OF THE INVENTION

The present invention is directed to the discovery that somenon-galloylated catechins will increase sensitization of MRSA againstselected antibiotics, where the selected antibiotic is already combinedwith a galloylated catechin.

In one aspect of the inventive subject matter, a method of reducing MIC(minimum inhibitory concentration) of an antibiotic drug in amethicillin resistant strain of S. aureus includes a step of exposingthe S. aureus to a galloylated catechin at a concentration effective toreduce the MIC of the antibiotic drug to a first level. In a furtherstep, it is recognized that application of a non-galloylated catechinsynergistically further decreases the MIC of the antibiotic drug fromthe first level to a second level, and in still another step, the S.aureus is exposed to the non-galloylated catechin in the presence of thegalloylated catechin at a concentration effective to reduce the MIC ofthe antibiotic drug to the second level.

Most preferably, the antibiotic drug may be characterized as abeta-lactam antibiotic (e.g., oxacillin, methicillin, penicillin), as atetracycline-type antibiotic, (e.g., tetracycline, quatrimycin,epitetracycline, etc.), and/or as a chloramphenicol-type antibiotic drug(e.g., chloramphenicol, dextramycin, 1-deoxychloramphenicol, etc.), thegalloylated catechin is ECG or EGCG, and/or the non-galloylated catechinis EC or EGC. Depending on the particular combination, it iscontemplated that the first level is equal or less than 50% of the MICof the antibiotic drug without exposing the Staphylococcus aureus to thegalloylated catechin, and/or the second level is equal or less than 5%of the MIC of the antibiotic drug without exposing the Staphylococcusaureus to the galloylated catechin.

In other contemplated aspects, the first level is equal or less than 5%of the MIC of the antibiotic drug without exposing the Staphylococcusaureus to the galloylated catechin, and the second level is equal orless than 0.5% of the MIC of the antibiotic drug without exposing theStaphylococcus aureus to the galloylated catechin. Therefore, in atleast some examples, the Staphylococcus aureus is exposed to thegalloylated catechin at a concentration of less than 10 microgram/ml,and to the non-galloylated catechin at a concentration of equal or lessthan 50 microgram/ml.

Viewed from a different perspective, the inventors also contemplate amethod of suppressing growth of a methicillin resistant strain ofStaphylococcus aureus (e.g., BB568, EMRSA-16, or EMRSA-15) wherein inone step Staphylococcus aureus is exposed to a combination of agalloylated catechin and an antibiotic drug, wherein the galloylatedcatechin and the antibiotic drug are present at a concentrationineffective to suppress growth. In another step, the Staphylococcusaureus is exposed to a non-galloylated catechin in the presence of thecombination at a concentration effective to suppress growth of theStaphylococcus aureus.

Similar to the method above, it is preferred that in such methods theantibiotic drug is a beta-lactam antibiotic at a concentration of 60microgram/ml, and wherein the galloylated catechin is ECG at aconcentration of 5 microgram/ml, and/or that the non-galloylatedcatechin (e.g., EC or EGC) is present at a concentration of at least 25microgram/ml, and more typically at least 50 microgram/ml.

Therefore, in another aspect of the inventive subject matter, anantibiotic drug (e.g., beta-lactam antibiotic) composition includes agalloylated catechin and a non-galloylated catechin, wherein thenon-galloylated catechin is present at a concentration tosynergistically reduce a MIC of the antibiotic drug with respect to thegalloylated catechin. Contemplated compositions further include aninformation associated with the composition that the galloylatedcatechin and the non-galloylated catechin are present in a synergisticcombination effective to reduce the MIC of the antibiotic drug withrespect to the galloylated catechin.

Most preferably, the galloylated catechin is ECG or EGCG, while thenon-galloylated catechin is EC or EGC. Typically, the non-galloylatedcatechin and the galloylated catechin are present in a weight ratio ofat least 3:1, and even more typically in a weight ratio of at least 6:1.While it should be appreciated that the composition may be formulated innumerous mariners, topical formulations (e.g., spray, cream, ointment,etc.) are particularly preferred.

Various objects, features, aspects and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting synergistic effects of the combination of ECand ECG on the MIC of oxacillin for EMRSA-16.

FIG. 2A is a table summarizing the effects of EC, EGC, and EGCG on thecapacity of ECG to sensitize selected S. aureus strains to oxacillin.

FIG. 2B is a table summarizing the effects of EC and EGC on the capacityof EGCG to sensitize S. aureus strains to oxacillin.

DETAILED DESCRIPTION

The inventors have unexpectedly discovered that catechin-mediatedsensitization of MRSA against various antibiotic drugs using galloylatedcatechins can further be enhanced by combining the galloylated catechinwith a non-galloylated catechin, and most preferably with thecorresponding non-galloylated catechin. Remarkably, the non-galloylatedcatechins that promote sensitization to an antibiotic drug appear tohave little or no effect on the minimum inhibitory concentration of thedrug when used by themselves.

As used herein, the terms “MRSA”, “methicillin resistant strain ofStaphylococcus aureus”, and “methicillin resistant Staphylococcusaureus” are used interchangeably herein and refer to S. aureus that isresistant to numerous, and more typically to all beta-lactam antibioticdrugs. Additional resistance may also be present with respect tocephalosporins and/or carbapenems. Hospital-associated MRSA isolatesoften are multiply resistant to other commonly used antimicrobialagents, including erythromycin, clindamycin, and tetracycline, whilecommunity-associated MRSA isolates are often resistant only tobeta-lactam antibiotic drugs and erythromycin. Resistance is confirmedfollowing protocols of the NCLS (National Committee for ClinicalLaboratory Standards), and is typically reflected by an MIC of equal orgreater than 4 microgram/ml in the Oxacillin MIC Test, a diffusion diskof equal or less than 10 mm or 19 mm in the Oxacillin Disk DiffusionTests or Cefoxitin Disk Diffusion Test, respectively. As also usedherein, the terms “minimum inhibitory concentration” and “MIC” are usedinterchangeably and refer to the minimum concentration of anantibacterial drug in a given culture medium below which bacterialgrowth is not inhibited.

The term “galloylated catechin” as also used herein refers to a compoundin which the C-ring of the compound (for nomenclature, see Formula 1below) is modified with a substituted or unsubstituted benzoic acidradical, and most typically a gallic acid ester radical. Most typically,the substituted or unsubstituted benzoic acid ester radical iscovalently bound to the C-ring in the 3-position. Contemplatedsubstituents on such substituted benzoic acids include one or morehydroxyl groups and their respective ester radicals, various alkyletherradicals, amino groups, nitro groups, sulfate and/or phosphate groups,each of which may be further substituted.

In contrast, the term “non-galloylated catechin” as used herein refersto a compound in which the C-ring may or may not be modified with asubstituent, but wherein that substituent, when present in the3-position of the C-ring, is not a gallic acid ester radical. Thus, andamong numerous other suitable substituents, the 3-position of the C-ringon non-galloylated catechins will be a hydrogen or hydroxy group.

In one preferred aspect of the inventive subject matter, the inventorsincubated strain EMRSA-16 of Staphylococcus aureus with varyingconcentrations of the galloylated catechin epicatechin gallate (ECG)between 0 mcg/ml and 12.5 mcg/ml to increase sensitivity of the strainagainst oxacillin. MICs were then recorded and the experiments wererepeated in the presence of the non-galloylated catechin epicatechin atvarying concentrations between 0 mcg/ml and 50 mcg/ml. Remarkably, theaddition of the non-galloylated catechin reduced the MIC in asignificant and synergistic manner. Similar results were obtained whenepicatechin was replaced with the non-galloylated epigallocatechin. Incontrast, when the galloylated epigallocatechin gallate was employed,substantially no significant and synergistic effect was obtained.

While not wishing to be bound by any particular theory or hypothesis,the inventors contemplate that nanomolar concentrations of catechins maybe able to modulate the structure and function of biological membranesthrough their capacity to partition into the phospholipid palisade.Galloylated catechins are thought to penetrate deeper intophosphatidylcholine and phosphatidylethanolamine bilayers than theirnon-galloylated analogues. Thus, it is expected that ECg and EGCg occupya deeper location in the membrane, and that EC and EGC localize at amore shallow location (closer to the phospholipid—water interface).These differences in membrane penetration parallel the capacity of thesemolecules to modify staphylococcal beta-lactam resistance.Interestingly, the quantities of ECg and EGCg that are incorporated intolipid bilayers are markedly increased in the presence of EC, raising thepossibility that the capacity of catechin gallates to reduce the levelof staphylococcal beta-lactam resistance could be potentiated bynon-galloylated catechins.

In other contemplated aspects of the inventive subject matter it shouldbe appreciated that numerous galloylated catechins other thanepicatechin gallate (ECG) are also deemed suitable for use herein. Amongother galloylated catechins, it is contemplated that appropriatecompounds include catechin gallate (CG), gallocatechin gallate (GCG),epigallocatechin gallate (EGCG), theaflavin monogallate A, theaflavinmonogallate B, and theaflavin digallate. It should further be noted thatthe catechins (galloylated and non-galloylated) contemplated hereininclude optical isomers, chiral centers, and/or stereoisomers, and thatall of such forms (and mixtures thereof) are contemplated herein.Similarly, non-galloylated catechins may include numerous alternativecompounds, including catechin (C), gallocatechin (GC), andepigallocatechin (EGC).

Therefore, preferred galloylated and non-galloylated catechinscontemplated herein will have a structure according to Formula 1

in which R₁, R₂, R₃, R₄, R₃′, R₄′, and R₅′ are independently H, OH, orM; wherein M is OC(O)R, OC(S)R, OC(NH)R, OR, or R, wherein R isoptionally substituted alkyl, alkenyl, alkynyl, alkaryl, or aryl (andmost preferably a mono-, di-, or trihydroxylated benzoic acid radical);wherein A, B, and C in the structure denote the respective rings; and

where the catechin is a galloylated catechin, R₃″ is a gallic acid esterradical; and

where the catechin is a non-galloylated catechin, R₃″ is M with theproviso that R is not a gallic acid radical in OC(O)R.

With respect to suitable concentrations of the galloylated catechin, itis generally contemplated that all concentrations are deemed appropriateso long as such concentration achieves at least some level ofsensitization (i.e., reduces the MIC of an antibiotic drug as comparedto growth under same conditions without galloylated catechin). Moreover,it should be recognized that different galloylated catechins willexhibit distinct reductions in MIC under comparable conditions. Stillfurther, a suitable concentration of galloylated catechins may also atleast in part be determined by a predetermined concentration ofnon-galloylated catechins. Therefore, various concentrations ofgalloylated catechins (including ECG) are contemplated and willgenerally be between 0 mcg/ml and 50 mg/ml (and even higher), but moretypically between 1 mcg/ml and 1 mg/ml, and most typically between 5mcg/ml and 100 mcg/ml.

Similarly, the concentration of contemplated non-galloylated catechinswill vary and depend on numerous factors, including the particularchemical structure of the non-galloylated catechin, the concentrationand type of the galloylated catechin, the bacterial strain, and/or theantibiotic drug. However, it is generally contemplated that theconcentration of the non-galloylated catechin is between 0 mcg/ml and 50mg/ml (and even higher), more typically between 1 mcg/ml and 1 mg/ml,and most typically between 5 mcg/ml and 100 mcg/ml. Furthermore, it ispreferred (but not necessarily so) that the non-galloylated catechin ispresent in at least equimolar amounts, more preferably in molar excessof 1.1-2.0 to 1, even more preferably in molar excess of 2.0-4.0 to 1,and most preferably in molar excess of 2.0-10.0 to 1 with respect to thegalloylated catechin.

Still further, it is preferred that the non-galloylated catechin is thecorresponding catechin of the galloylated catechin. For example, wheresensitization to a first level is effected by ECG, it is preferred thatthe non-galloylated catechin is EC. In another example, wheresensitization to a first level is effected by EGCG, it is preferred thatthe non-galloylated catechin is EGC. However, it should also beappreciated that more than one catechin (galloylated and/ornon-galloylated) may be employed in contemplated combinations. Forexample, sensitization to a first level may be effected by a mixture ofcatechins that includes at least one galloylated catechin, and thesensitization to a second, lower level may then be effected by a singlenon-galloylated catechin. On the other hand, and where desirable,sensitization to a first level may be effected by a single catechin, andthe sensitization to a second, lower level may then be effected by amixture that includes at least one non-galloylated catechin.

Where catechin mixtures are employed, it is particularly preferred thatthe mixture is isolated from a plant (e.g., tea plant, grape, blueberry,etc.). Such catechin isolates may be normalized to a specificformulation in which the ratio of galloylated to non-galloylatedcatechins is predetermined. For example, a typical percentage ofindividual catechins in a green tea extract with a dominatinggalloylated catechin proportion may be 10-15% EGCg, 2-3% ECG, 2% EC, and2-3% EGC (e.g., Suganuma et al., 1999, Can. Res. 59:44-47). On the otherhand, extracts may be prepared or modified to have predominantlynon-galloylated catechins (e.g., with 10-20% of EGC and EC, 2-3% ECG,and 5% EGCG). In addition, caffeine, theobromine, theophylline, andphenolic acids, such as gallic acid, may also be present as constituentsof green tea extracts, and are typically present in smaller quantitiesthan the polyphenols. Among other contemplated extracts, particularlysuitable polyphenol extracts include polyphenon E and polyphenon B, bothof which are commercially available from Mitsui Norin Japan (1-2-9Nishishinbashi, Minato-ku, Tokyo, 105-8427, Japan), and which may beemployed as further base material for specific preparations.

With respect to the antibiotic drugs, it is generally contemplated thatnon-galloylated catechins may further increase sensitivity (i.e., reduceMIC) of previously sensitized MRSA to numerous antibiotic drugs,including all or almost all beta-lactam antibiotics, tetracyclines,and/or chloramphenicol-type drugs. Thus, and among other antibioticdrugs, contemplated antibiotics include various penicillins (e.g.,benzathine penicillin, benzylpenicillin (penicillin G),phenoxymethylpenicillin (penicillin V), dicloxacillin, flucloxacillin,amoxicillin, ampicillin, etc.), various cephalosporins (e.g.,cephalexin, cephalothin, cephazolin, cefuroxime, cefamandole,ceftriaxone, cefotaxime, etc.), various carbapenems (e.g., imipenem,meropenem, ertapenem, etc.), monobactams (e.g., aztreonam), varioustetracyclins (e.g., tetracycline, quatrimycin, epitetracycline, etc.),and various chloramphenicol-type antibiotic drugs (e.g.,chloramphenicol, dextramycin, 1-deoxychloramphenicol, etc.).

Depending on the type of antibiotic drug and concentration ofgalloylated catechin, it should be appreciated that the increase insensitivity using the non-galloylated catechin may vary considerably.However, it is generally contemplated that the increase in sensitivity(i.e., reduction in MIC) is at least 2-fold (e.g., from 20 mcg/ml to 10mcg/ml), more typically at least 3-fold, even more typically 4-6 fold,and most typically at least 5-10 fold (e.g., from 50 mcg/ml to 10mcg/ml).

It is still further contemplated that exposure of the MRSA to thegalloylated and/or non-galloylated catechin is preferably, but notnecessarily performed in the presence of the antibiotic drug. Mosttypically (but again not necessarily), the galloylated andnon-galloylated catechin are administered together to the environment inwhich the MRSA is present. For example, and especially where theadministration is ex vivo or a topical administration (e.g.,disinfectant spray, in vitro culture, topical ointment, etc.), thecatechin combination may be admixed to the antibiotic solution. Inanother example, and especially where the combination is administered toa mammal, the catechin combination may also be administered orally andseparately from the antibiotic preparation (which may or may not beadministered orally or parenterally).

Therefore, it should be recognized that the galloylated and/ornon-galloylated catechin may be administered in numerous formulations,and especially preferred formulations include oral formulations in solid(e.g., isolated catechin, or polyphenol powder from tea extracts) orliquid form (e.g., liquid catechin preparation or tea extract). Furtherparticularly preferred formulations include topical formulations (e.g.,as cream, ointment, gel, lotion, etc.). There are numerous suchcommercially available formulations known in the art, and all of thoseare deemed suitable for use herein. The concentration of the galloylatedand/or non-galloylated catechin in such formulations will typically besuch that application in a single dosage unit will result in a localconcentration of less than 1 mg/ml.

Based on these and further considerations, the inventors thereforecontemplate a method of reducing MIC of an antibiotic drug in MRSA inwhich the Staphylococcus aureus is exposed to a galloylated catechin ata concentration effective to reduce the MIC of the antibiotic drug to afirst level. In another step, it is recognized (e.g., via written ordisplayed information) that application of a non-galloylated catechinfurther synergistically decreases the MIC of the antibiotic drug fromthe first level to a second level, and in still another step, theStaphylococcus aureus is exposed to the non-galloylated catechin in thepresence of the galloylated catechin at a concentration effective toreduce the MIC of the antibiotic drug to the second level. For example,in a typical application, suitable first levels are equal or less than50% of the MIC of the antibiotic drug without exposing theStaphylococcus aureus to the galloylated catechin, and suitable secondlevels are equal or less than 5% of the MIC of the antibiotic drugwithout exposing the Staphylococcus aureus to the galloylated catechin.In such examples, the inventors discovered that the MRSA was exposed tothe galloylated catechin at a concentration of less than 10 microgram/mland to the non-galloylated catechin at a concentration of equal or lessthan 50 microgram/ml. In other examples, the inventors found that thefirst level was equal or less than 5% of the MIC and that the secondlevel was equal or less than 0.5% of the MIC.

Therefore, suitable methods also include methods of suppressing growthof MRSA in which the MRSA is exposed to a combination of a galloylatedcatechin and an antibiotic drug, wherein the galloylated catechin andthe antibiotic drug are present at a concentration (e.g., ECG at aconcentration of 5 microgram/ml, and beta-lactam antibiotic at aconcentration of 60 microgram/ml) ineffective to suppress growth. Inanother step, the MRSA is then exposed to a non-galloylated catechin(e.g., EC or EGC at a concentration of at least 25 mcg/ml, and moretypically at least 50 mcg/ml) in the presence of the combination at aconcentration effective to suppress growth of the MRSA. Such methods areparticularly desirable to convert an MRSA (e.g., BB568, EMRSA-16, andEMRSA-15) from a resistant or intermediate character (with respect to aparticular antibiotic drug) to a sensitive character.

In still further contemplated aspects, drug compositions are thuscontemplated that include a galloylated catechin (e.g., ECG or EGCG), anon-galloylated catechin (EC or EGC), and optionally an antibiotic drug(e.g., a beat-lactam antibiotic), wherein the non-galloylated catechinis present at a concentration to synergistically reduce a minimuminhibitory concentration of the antibiotic drug with respect to thegalloylated catechin. Such compositions are typically further associatedwith an information that the galloylated catechin and thenon-galloylated catechin are present in a synergistic combination thatreduces the MIC of the antibiotic drug with respect to the galloylatedcatechin. Among other suitable ratios, the non-galloylated catechin andthe galloylated catechin are present in a weight ratio of at least 1:1,more typically at least 3:1, and even more typically at least 6:1. Asalready discussed above, it should be recognized that the compositionmay be formulated in numerous manners, including oral and topicalformulations. However, it is particularly preferred that the formulationis a topical formulation.

EXPERIMENTS Enhanced Binding of ECG to Membranes Mediated By EC

As S. aureus membranes are uniquely composed of phosphatidylglycerol(63-74%), lysylphosphatidylglycerol (17-22%), and cardiolipin (5-15%),the inventors determined the capacity of non-galloylated catechins toenhance ECG binding to S. aureus using the HPLC assay described byKajiya et al (Kajiya, K., S. Kumazawa, and T. Nakayama. 2001. Stericeffects on the interaction of tea catechins with lipid bilayers. Biosci.Biotechnol. Biochem. 65:2638-2643). EC, EGC, ECG and EGCG were providedby the Tokyo Food Techno Co. Ltd., Tokyo, Japan. S. aureus BB568 is aconstitutive PBP2a producer (provided by B. Berger-Bächi, University ofZürich), and EMRSA-15 and EMRSA-16 were clinical isolates from the RoyalFree Hospital, London. Binding of ECG to mid-logarithmic EMRSA-16 cellswas enhanced by EC at concentrations of 25 mcg/ml. 13% of the EC poolbound after 20 min incubation at 35° C., and 22% of ECG was associatedwith the cells. In the presence of EC, ECG binding rose to 41%. ECbinding was also enhanced by the presence of ECG (35.5%). EC appears,therefore, to facilitate ECG binding to staphylococcal cells as well asto PC and PE liposomes.

Sensitization Test with S. aureus

To investigate if this cooperative binding elicited enhanced biologicalactivity, the capacity of EC and EGC to increase the degree ofsensitization of S. aureus to oxacillin was determined. Checkerboard MICassays were performed in 96-well microtiter trays with an inoculum ofabout 10⁴ CFU in 200 μl of Mueller-Hinton broth (Oxoid, Basingstoke,United Kingdom) supplemented with 2% NaCl. MIC values were obtainedafter incubation at 35° C. for 24 h. S. aureus ATCC29213 was used as thestandard susceptible strain. Fractional inhibitory concentration (FIC)indices for triple combinations were calculated as follows:ΣFIC=FIC_(OXA)+FIC_(B)+FIC_(C)=[C^(comb) _(OXA)/MIC^(OXA)]+[C^(comb)_(B)/MIC_(B)]+[C^(comb) _(C)/MIC_(C)], where C^(comb) _(B), and C^(comb)_(c) are the concentrations of catechins tested. C^(comb) _(OXA) is thelowest concentration of oxacillin in the combination that inhibitedgrowth, and MIC_(OXA), MIC_(B) and MIC_(C) are the MICs of the compoundswhen used alone. For combinations of two compounds the term (C^(comb)_(C)/MIC_(C)) was omitted. An FIC index of ≦0.5 indicates synergy.

We determined the effect of EC, EGC and EGCG on the capacity of ECG tosensitize EMRSA-16, EMRSA-15 and BB568 to oxacillin, and the results forthese experiments are listed in FIG. 2A. The MICs of oxacillin forBB568, EMRSA-16 and EMRSA-15 were 256, 512 and 16 mg/ml, respectively.The catechin compounds had little or no intrinsic anti-staphylococcalactivity, with MICs ranging from 64-512 mg/ml. An ECG concentration of12.5 mg/ml reduced the oxacillin MIC for S. aureus BB568 and EMRSA-16 tobelow the oxacillin breakpoint; a similar effect was achieved forEMRSA-15 with the lower concentration of 3.12 mg/ml. Remarkably, thenon-galloylated catechins EC and EGC were unable to reduce the MIC ofoxacillin against these isolates. However, these compounds markedlyenhanced the capacity of ECG to reduce oxacillin resistance. Aconcentration of 3.12 mg/ml of ECG reduced the MICs to 64 and 128 mg/mlfor BB568 and EMRSA-16, respectively. In combination with 6.25 mg/ml ofEC, oxacillin susceptibility increased to 8 mg/ml for these isolates;increasing the EC concentration to 25 mg/ml produced values of 2 mg/mlin the presence of 3.12 mg/ml of ECG. Similar reductions were observedwhen EGC was used in combination with ECG. FIC indices indicated strongsynergy between oxacillin, ECG and either EC or EGC against all three S.aureus isolates. At high concentrations, a combination of ECG (≧12.5mg/ml) with both EC and EGC (≧50 mg/ml) inhibited the growth of EMRSA-16in the absence of oxacillin.

The enhancement by EC and EGC of ECG-mediated sensitization to oxacillinwas clearly concentration dependent, as illustrated in FIG. 1 (depictingthe effect of ECG and EC on oxacillin MICs for EMRSA-16). Thegalloylated catechin EGCG was significantly less effective at reducingthe MIC for oxacillin in the presence of ECG. For example, with 3.12mg/ml of ECG, 25 mg/ml of EGCG reduced the MIC only twofold. At thehigher ECG concentration of 12.5 mg/ml, EGCG compromised the capacity ofECG to reduce the MICs for strains BB568 and EMRSA-16. EGCG was alsoable to sensitize BB568, EMRSA-16 and EMRSA-15 to oxacillin (see FIG.2B) but the effect was much less pronounced than that associated withECG. Both EC and EGC could enhance sensitization (as shown in FIG. 2B),but the affect was correspondingly less in comparison to[EC/EGC-ECG-oxacillin] combinations, as shown in FIG. 2A.

The inventors therefore contemplate that the capacity of non-galloylatedcatechins such as EC and EGC enhance the oxacillin susceptibility ofMRSA strains by the galloyl catechins ECG and EGCG via targeting of thestaphylococcal cytoplasmic membrane. Such hypothesis would be confirmedby the increased ECG membrane binding in the presence of EC or EGC ifthere was a causal connection between membrane binding and sensitivity.In further support of such hypothesis is the fact that catechinsgenerally do not enter cells, which makes it more likely that they maymodulate beta-lactam resistance in S. aureus by alteration ofbiophysical properties of the membrane (e.g., by compromising thefunction of proteins associated with the bilayer and thus affectingtransport of materials across the membrane).

The above data clearly demonstrate the significant and synergisticcharacter of the interaction between galloylated and non-galloylatedcatechins in the presence of an antibiotic with regard to theirantimicrobial effect. Such finding is particularly surprising asprevious studies have suggested that the gallate group is essential forepicatechin gallate activity (Stapleton, P. D., S. Shah, J. C. Anderson,Y. Hara, J. M. T. Hamilton-Miller, and P. W. Taylor. 2004. Modulation ofbeta-lactam resistance in Staphylococcus aureus by catechins andgallates. International J. Antimicrol. Agents 23:462-467). Furthermore,it is unlikely that the non-galloylated catechins act as an esteraseinhibitor as these compounds lack an ester group. In addition,esterase-stable derivatives of ECG have similar activities when used incombination with oxacillin against S. aureus, suggesting esteraseactivity is not a critical factor in vitro (Anderson, J. C., C. Headley,P. D. Stapleton, and P. W. Taylor. 2005. Synthesis and antibacterialactivity of a hydrolytically stable (−)-epicatechin gallate analogue forthe modulation of beta-lactam resistance in Staphylococcus aureus.Bioorg. Med. Chem. Lett. 15:2633-2635). However, it is noted that otherforms of inactivation, and particularly modification to the catechinmoiety, where EC and EGC could act as potential inhibitors, cannot beruled out.

Thus, specific embodiments and applications of compositions and methodsof sensitizing MRSA to oxacillin have been disclosed. It should beapparent, however, to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Furthermore, where a definition or use of a termin a reference, which is incorporated by reference herein isinconsistent or contrary to the definition of that term provided herein,the definition of that term provided herein applies and the definitionof that term in the reference does not apply.

1. An antibiotic drug composition, comprising: a galloylated catechin, anon-galloylated catechin, and an antibiotic drug, wherein the antibioticdrug is a beta-lactam antibiotic; wherein the non-galloylated catechinis present at a concentration to synergistically reduce a minimuminhibitory concentration of the antibiotic drug with respect to thegalloylated catechin, and wherein the non-galloylated catechin and thegalloylated catechin are present in a weight ratio of at least 3:1; andan information associated with the composition that the galloylatedcatechin and the non-galloylated catechin are present in a synergisticcombination that reduces the minimum inhibitory concentration of theantibiotic drug with respect to the galloylated catechin.
 2. Theantibiotic drug composition of claim 1 wherein the galloylated catechinis ECG or EGCG.
 3. The antibiotic drug composition of claim 1 whereinthe non-galloylated catechin is EC or EGC.
 4. The antibiotic drugcomposition of claim 1 wherein the non-galloylated catechin and thegalloylated catechin are present in a weight ratio of at least 6:1. 5.The antibiotic drug composition of claim 1 wherein the antibiotic drugis a beta-lactam antibiotic is selected from the group consisting of anoxacillin, methicillin, and a penicillin.
 6. The antibiotic drugcomposition of claim 1 wherein the composition is formulated as atopical composition.
 7. A method of suppressing growth of a methicillinresistant strain of Staphylococcus aureus, comprising: exposing theStaphylococcus aureus to a combination of a galloylated catechin and anantibiotic drug, wherein the antibiotic drug is a beta-lactamantibiotic, wherein the galloylated catechin and the antibiotic drug arepresent at a concentration ineffective to suppress growth; and exposingthe Staphylococcus aureus to a non-galloylated catechin in the presenceof the combination at a concentration effective to suppress growth ofthe Staphylococcus aureus, wherein the non-galloylated catechin and thegalloylated catechin are present in a weight ratio of at least 3:1. 8.The method of claim 7 wherein the antibiotic drug is at a concentrationof 60 microgram/ml, and wherein the galloylated catechin is ECG at aconcentration of 5 microgram/ml.
 9. The method of claim 8 wherein thenon-galloylated catechin is EC or EGC at a concentration of at least 25microgram/ml.
 10. The method of claim 7 wherein the non-galloylatedcatechin is EC or EGC at a concentration of at least 50 microgram/ml.11. The method of claim 7 wherein the Staphylococcus aureus is selectedfrom the group consisting of BB568, EMRSA-16, and EMRSA-15.